Present address: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
Assembly of the Yersinia injectisome: the missing pieces
Article first published online: 13 JUL 2012
© 2012 Blackwell Publishing Ltd
Volume 85, Issue 5, pages 878–892, September 2012
How to Cite
Diepold, A., Wiesand, U., Amstutz, M. and Cornelis, G. R. (2012), Assembly of the Yersinia injectisome: the missing pieces. Molecular Microbiology, 85: 878–892. doi: 10.1111/j.1365-2958.2012.08146.x
- Issue published online: 26 AUG 2012
- Article first published online: 13 JUL 2012
- Accepted 19 June, 2012.
- Top of page
- Experimental procedures
- Supporting Information
The assembly of the type III secretion injectisome culminates in the formation of the needle. In Yersinia, this step requires not only the needle subunit (YscF), but also the small components YscI, YscO, YscX and YscY. We found that these elements act after the completion of the transmembrane export apparatus. YscX and YscY co-purified with the export apparatus protein YscV, even in the absence of any other protein. YscY-EGFP formed fluorescent spots, suggesting its presence in multiple copies. YscO and YscX were required for export of the early substrates YscF, YscI and YscP, but were only exported themselves after the substrate specificity switch had occurred. Unlike its flagellar homologue FliJ, YscO was not required for the assembly of the ATPase YscN. Finally, we investigated the role of the small proteins in export across the inner membrane. No export of the reporter substrate YscP1–137-PhoA into the periplasm was observed in absence of YscI, YscO or YscX, confirming that these proteins are required for export of the first substrates. In contrast, YscP1–137-PhoA accumulated in the periplasm in the absence of YscF, suggesting that YscF is not required for the function of the export apparatus, but that its polymerization opens the secretin YscC.
- Top of page
- Experimental procedures
- Supporting Information
Various bacteria use type III secretion (T3S) systems to inject effector proteins into host cells. This leads either to mutual benefit or to pathogenesis (Cornelis and Wolf-Watz, 1997; Galan and Collmer, 1999). Targets and biochemical activities of these effectors widely vary between bacterial species. The most frequent targets are cellular signalling pathways and actin cytoskeleton dynamics (Mota and Cornelis, 2005). While this high degree of diversity among the effectors allows adaptation to the specific lifestyle of the bacterium, the apparatus itself, also called injectisome, is conserved among species (Cornelis, 2006). The injectisome is evolutionary related to the bacterial flagellum (Fields et al., 1994; Woestyn et al., 1994; Van Gijsegem et al., 1995; Kubori et al., 1998). The similarity resides on the part which spans both bacterial membranes and includes the protein export apparatus itself. In the flagellum, this part is completed by two proteins which make it rotate by harnessing the proton motive force (Larsen et al., 1974). On the extracellular side, the flagellum possesses a filament that translates its rotation into propelling force, while the injectisome has an extracellular needle (Kubori et al., 1998; Blocker et al., 1999; Sani et al., 2007) that bridges the gap between bacterium and host cell to allow the direct translocation of effector proteins (Mota et al., 2005; West et al., 2005).
The basal part of the injectisome is made of more than 10 different proteins in various copy numbers and has a molecular weight of more than a mega-Dalton. It consists of a structurally well-characterized pair of rings spanning the two membranes (YscCDJ in Yersinia) (Spreter et al., 2009), a less understood export apparatus and a cytosolic part. Five transmembrane proteins (YscRSTUV) (Plano et al., 1991; Allaoui et al., 1994; Fields et al., 1994) form the export apparatus within the ring spanning the inner membrane (IM) (MS ring); the cytosolic part of the basal body includes a multimeric protein (YscQ) that shares homology to the flagellar C ring (cytosolic) components, a hexameric ATPase (YscN) and two ancillary proteins (YscKL). Recent data suggest that in Salmonella, a cytosolic complex of the C ring homologue and the two ancillary proteins acts as a sorting platform for T3S cargo proteins (Lara-Tejero et al., 2011). Figure 1 shows a schematic representation of the injectisome; Table 1 lists the main components and their homologues between T3S systems.
|Protein(s)||Function||Stoichiometry||Yersinia||Salmonella SPI-1||Shigella||Pseudomonas syringae||Flagellum|
|Hydrophobic translocators||Pore in host cell||15–20 in total||YopB||SipB||IpaB||HrpK||–|
|Hydrophilic translocator||Scaffold for pore formation||5||LcrV||SipD||IpaD||–||–|
|Needle||Gap space between bacterium and host||About 140||YscF||PrgI||MxiH||HrpA||–|
|Inner rod||Extension of channel within basal body||Unknown||YscI||PrgJ||MxiI||HrpB2||FlgB + -C + -F + -G|
|OM secretin ring||Penetration of OM, PG anchoring||12–15||YscC||InvG||MxiD||HrcC||–|
|MS ring||Penetration of IM, connection to OM||12 or 24 of each protein||YscD||PrgH||MxiG||HrcD||FliG|
|IM export apparatus||Substrate selection and export||Unknown, multiple copies of YscV||YscR||SpaP||Spa24||HrcR||FliP|
|ATPase||Chaperone detachment, export energization||6 or 12||YscN||InvC/SpaL||Spa47||HrcN||FliI|
|C ring||Substrate selection||Unknown||YscQ||InvK/SpaO||Spa33||HrcQa||FliN + FliM (+ FliG)|
|Interactors of ATPase/C ring||ATPase regulation, interaction platform?||Unknown||YscL||OrgB||MxiN||HrpE||FliH|
|Ruler||Needle length control, substrate specificity switch||n.a.||YscP||InvJ||Spa32||HrpP||FliK|
Most T3S effectors and some machinery components require the assistance of small acidic cytosolic proteins, called T3S chaperones, for their function (Wattiau and Cornelis, 1993; Wattiau et al., 1996; Page and Parsot, 2002). Often, these chaperones are encoded next to their cargo proteins. Effector proteins are bound by class I chaperones; class II chaperones bind the two hydrophobic translocator proteins. Class III chaperones, mainly known from studies on the flagellum, prevent premature polymerization within the bacterium of components of the distal structures of the apparatus, like the cap protein or flagellin. The T3S needle subunit is bound by two class III chaperones (YscE and YscG in Yersinia) (Day et al., 2000; Quinaud et al., 2005; 2007; Sun et al., 2008; Ple et al., 2010).
Studies of different T3S systems consistently showed that the two membrane-spanning rings of the basal body can assemble independently of any other T3S component (Kimbrough and Miller, 2000; 2002; Kubori et al., 2000; Sukhan et al., 2001; Ogino et al., 2006; Diepold et al., 2010). In Salmonella SPI-1, the MS ring components PrgH and PrgK were shown to form a somewhat stable ring structure (Kimbrough and Miller, 2000; 2002; Schraidt et al., 2010), suggesting that assembly starts at the MS ring. In Yersinia the YscC secretin ring in the outer membrane (OM) has been shown to be a stable structure (Koster et al., 1997; Burghout et al., 2004a,b) and assembly of the Yersinia injectisome was found to nucleate at the OM secretin, from where it progresses stepwise inwards to the MS ring and the cytosolic components, ATPase and C ring (Diepold et al., 2010).
More recent studies revealed that assembly of the IM export apparatus can occur independently (Wagner et al., 2010; Diepold et al., 2011). Wagner et al. showed that the Salmonella SPI-1 export machinery proteins not only can form a complex on their own, but also that this complex can form the starting point of complete injectisomes (Wagner et al., 2010). Similarly, in Yersinia, the export apparatus can assemble independently, but requires the membrane rings for stable anchoring within the membrane (Diepold et al., 2011). This is consistent with recent data from the flagellum, where the IM export apparatus component FlhA was found to stabilize and promote multimerization of the MS ring subunit FliF (Li and Sourjik, 2011). Thus, assembly starts independently at the export apparatus and at the secretin ring. Both assembly branches then converge, which anchors the IM export apparatus within the peptidoglycan layer.
Formation of the needle is the next observable step in the assembly. The export of the subunits is accomplished by the nascent injectisome itself. The export apparatus is capable of exporting sequentially different classes of substrates. The ‘early’ substrates include the needle subunit YscF, the tip component that resides at its distal end (Mueller et al., 2005), and the molecular ruler YscP, which switches the substrate specificity after the needle has reached its programmed length (Minamino et al., 1999; Agrain et al., 2005a). After the T3S export apparatus has exported the needle and needle tip subunits, the injectisome is fully assembled and ready for export of the hydrophobic translocators and effectors. However, this export is only triggered by the change from ‘secretion-restrictive’ to ‘secretion-permissive’ conditions: in vivo through contact with a eukaryotic cell or, in vitro, by incubation in the absence of Ca2+ (Pettersson et al., 1996; Cornelis and Wolf-Watz, 1997). In Yersinia, besides the needle subunit YscF and the translocators at the tip of the needle (see Table 1), three proteins that are exported themselves, YscI, YscO and YscX, are essential for the final translocation of the effectors. YscI and YscO have been shown to be already required for the assembly of the needle, like related small proteins from other T3S systems (Payne and Straley, 1998; Kimbrough and Miller, 2000). YscI resembles MxiI (Shigella) and PrgJ (Salmonella) that have been assigned to form a rod structure extending the hollow needle at the centre of the basal body (Sukhan et al., 2003; Marlovits et al., 2004; Sal-Man et al., 2012). Gene synteny and similarities in size and secondary structure link YscO to InvI (Salmonella), Spa13 (Shigella) and FliJ (flagellum). A recent structural analysis has suggested that FliJ has similarity to the γ-subunit of the F0F1-ATPase, which is part of the central stalk (Ibuki et al., 2011). In addition, FliJ was shown to promote in vitro the hexamerization of the FliI ATPase (Ibuki et al., 2011). However, FliJ has also been described as a flagellar chaperone escort protein whose function is to recruit unloaded chaperones for the minor filament-class subunits of the filament cap and hook–filament junction substructures (Evans et al., 2006). Like FliJ, YscO has also been shown to bind to some T3S chaperones (Evans and Hughes, 2009). Whether YscO is indeed a component of the ATPase or a chaperone escort, the reason why it is secreted is unclear. Finally, YscX (Iriarte and Cornelis, 1999) is a 13.6 kDa protein encoded besides YscY, a non-secreted protein which binds to YscX like a chaperone (Day and Plano, 2000). Both proteins are essential for the function of the injectisome (Iriarte and Cornelis, 1999), but not for the assembly of the C ring (Diepold et al., 2010). YscY interacts also with SycD (LcrH), the chaperone of translocators, which suggests that it also participates in global regulation of the system (Broms et al., 2005). The function of YscX is completely unknown.
In this study, we have analysed the assembly and physiological role of a set of small soluble proteins that are required before the T3S apparatus can become functional. We found that YscX and its proposed chaperone YscY directly and tightly bind to YscV, an export apparatus protein that we have recently shown to be present in multiple copies at the basal body. We show that the addition of YscX and YscY to YscV, together with the recruitment of YscO, allows the export of YscF and YscI, the two first proteins to be exported. YscI, YscO and YscX are required for the export of the reporter substrate YscP1–137-PhoA across the IM, while YscF is not. However, in the absence of YscF, the reporter substrate is exported to the periplasm, indicating that it is YscF that opens the YscC secretin ring.
- Top of page
- Experimental procedures
- Supporting Information
YscX and YscY strongly and directly bind to the cytosolic domain of YscV
To get more insight into the relation between the export apparatus and the cytosolic part of the injectisome, we expressed yscV-his-flag in trans in a yscV mutant strain. This fusion protein has previously been shown to be functional, as it restored the Ca2+-controlled secretion of effector proteins (Diepold et al., 2011). We purified YscV-His-FLAG and analysed the co-purified proteins by mass spectrometry. A mock purification was carried out in parallel with bacteria expressing untagged YscV, and all the hits obtained from this purification were purged from the list of YscV-His-FLAG-binding proteins. The results confirmed the association of YscV with the membrane ring proteins YscC, -D, -J, and the needle subunit YscF, which had been previously reported (Wagner et al., 2010; Diepold et al., 2011). However, to our surprise the strongest hits (except for YscV itself) were not the co-purified machinery components, but the two cytosolic proteins YscX and YscY. Since an antibody against YscX was available, the presence of YscX in the eluate was confirmed by an immunoblot analysis (Fig. 2A). Consistent with their proposed cytosolic localization, YscX and YscY did not bind to YscV deprived of its C-terminal cytosolic part in a parallel co-immunoprecipitation with the tagged N-terminal transmembrane part of YscV (YscVTM-His-FLAG) (Table 2). YscX and YscY required each other for co-purification with YscV, but no other tested export machinery protein (Fig. 2B, Table 2).
|YscV (neg. ctrl.)||YscV(TM)-His-FLAG||YscV-His-FLAG|
To test if YscX, YscY and the cytosolic domain of YscV (YscVC) directly interact with each other, we coexpressed the three proteins in Escherichia coli. YscX and YscY co-purified with the N-terminally GST-tagged YscVC, even without additional cross-linking (Fig. 2C). Analysis of truncated versions of YscVC revealed that the presence of all structural domains (SD) of YscVC (Bange et al., 2010; Lilic et al., 2010; Moore and Jia, 2010; Worrall et al., 2010) was required for significant co-purification of YscX and YscY (Fig. 2C and D, Fig. S1): The two GST-YscVC constructs comprising all SDs (lanes 4 and 5 in Fig. 2C) co-purified with substantial amounts of two proteins corresponding by size to YscX and YscY (red and green stars); we confirmed the identity of YscX by immunoblot (Fig. S1). In contrast, truncations of the C-terminal structural domains of YscVC (lanes 1–3) were expressed, soluble and could be purified, but did not pull down YscX and YscY. The constructs missing the N-terminal part or the complete SD1 (lanes 6 and 7), SD1+2, or SD4 (not shown in Fig. 2) were insoluble and could therefore not be analysed.
YscY-EGFP forms fluorescent spots, suggesting its presence in multiple copies at the injectisome
Previously, we have shown that YscV forms an oligomer, of so far unknown stoichiometry, within the injectisome (Diepold et al., 2011). Therefore, YscX and YscY might be present in multiple copies as well. In this case, fluorescently labelled YscX and YscY would form spots like YscV-EGFP (Diepold et al., 2011). We fused egfp to both termini of yscX and yscY and tested these hybrid constructs for trans-complementation of yscX and yscY deletion mutants. Consistent with previous results (Day and Plano, 2000; Yang et al., 2007), the fusions to YscX were unstable and could not complement a yscX strain for effector secretion. However, YscY-EGFP complemented the respective mutant in trans and was consequently analysed in more detail. To prevent effects of overexpression, yscY was substituted by the yscY-egfp allele on the pYV virulence plasmid by homologous recombination. YscY-EGFP was stable and functional (Fig. 3A, Fig. S2) and, after induction of the T3S system, formed spots at the cell periphery (Fig. 3B). This strongly suggested that YscY is present in multiple copies at the cytosolic side of the injectisome. Fluorescence intensity of the YscY-EGFP and YscV-EGFP was comparable, compatible with stoichiometric binding (Fig. 3B).
Since we have observed that YscY binds to YscV and that this binding requires the presence of YscX, we combined the yscY-egfp allele with deletions of yscV and yscX and monitored the formation of fluorescent spots. Consistent with our previous results, YscY-EGFP did not form spots in strains lacking either YscV or YscX (Fig. 3B). These results show that the YscY-EGFP spots reflect the assembly of YscY-EGFP onto YscV and they confirm that this interaction requires YscX.
Since YscX–YscY directly bind to YscV, and the assembly of YscV depends neither on the presence of the cytosolic components YscN (ATPase) and YscQ (C ring), nor on any of the small soluble T3S proteins YscF, YscI or YscO (Diepold et al., 2011), we infer that the assembly of YscY is unlikely to require any of these factors. Exemplarily, we tested this for the ATPase YscN and found that, indeed, YscY-EGFP forms spots in the absence of YscN (Fig. 3B).
We finally investigated whether the assembly of YscY is coupled to the secretion state of the injectisome. For this purpose, we monitored the formation of YscY-EGFP spots in bacteria incubated with and without Ca2+. As shown in Fig. 3B, YscY-EGFP formed spots irrespective of the secretion state. The intensity of the spots was weaker in the presence of Ca2+, corresponding to the lower expression level of YscY-EGFP in secretion-restrictive conditions (Fig. S2). The same was previously observed with other T3S components (Diepold et al., 2010); the reason of this is presently unknown.
Since YscX and YscY directly bind to YscV, they might assist the assembly or multimerization of YscV itself. We thus tested whether YscV-EGFP forms spots in the absence of YscX or YscY. YscV did assemble normally in these cases (Fig. 4A), indicating that YscX and YscY are not involved in assembly of the export apparatus or its connection to the membrane rings.
To test whether YscX and YscY are needed for needle assembly, we next tried to purify needles of bacteria lacking either of the two components. As shown in Fig. 4B, the presence of both proteins was required for needle formation. In conclusion, YscX and YscY seem to be required for the completion of the export apparatus, making it ready for the export of the early substrates.
YscI, YscO, YscX and YscY are required for ‘basic export’ across the IM, whereas YscF opens the secretin channel
Besides YscX and YscY, other small early substrates, namely YscF, YscI and YscO, are known to be essential for T3S (Allaoui et al., 1995; Payne and Straley, 1998; Iriarte and Cornelis, 1999), but not for the assembly of the basal body (Diepold et al., 2010; 2011). Thus, they probably all play a late role in the assembly of the injectisome. However, it is possible that the export machinery at the IM is already functional in the absence of one or more of these early substrates, but that the substrates accumulate in the periplasm. Therefore, we measured the activity of alkaline phosphatase (PhoA), deprived of its own signal sequence and fused to the N-terminal export signal sequences of YscP (YscP1–137) (Agrain et al., 2005b), as a reporter substrate. PhoA is active in the periplasm and inactive in the cytosol (Michaelis et al., 1983; San Millan et al., 1989). Hence, hybrids between PhoA and classical T3S substrates are not expected to display strong phosphatase activity, unless they accumulate in the periplasm because of a defect in the apparatus. Consistent with this, in wild-type bacteria, the phosphatase activity remained low despite the presence of PhoA in the supernatant (Fig. 5). In the absence of the structural injectisome components YscC, -D, -J, or of the IM export apparatus components YscR, -S, -T, -U, -V, neither activity nor export into the supernatant of PhoA could be detected (data not shown). However, in the absence of the needle component YscF, YscP1–137-PhoA gave a significant PhoA activity, even though no PhoA could be detected in the supernatant (Fig. 5). PhoA without a T3S export signal was neither exported nor active (data not shown). Our interpretation is that, in yscF mutants, the basic export machinery is active but can only translocate substrates to the periplasm, which would imply that it is the needle that opens the secretin ring in the OM. In contrast, in the absence of YscI, YscO, YscX or YscY, no PhoA activity could be detected (Fig. 5). We infer from this that YscI, YscO, YscX and YscY, but not YscF, are already required for the ‘basic export function’ of the IM export machinery.
Role of YscO in assembly
While YscF forms the needle, it is assumed that YscI could form the rod, and YscX and YscY bind to YscV, the role of YscO is still elusive. Since the YscO homologue FliJ has been proposed to assist the multimerization of the ATPase (Ibuki et al., 2011), we tested if YscO actually promotes the assembly of the ATPase YscN to the injectisome. Therefore, EGFP-YscN (Diepold et al., 2010) was expressed in trans in a non-polar yscNO double-deletion strain and in a single yscN deletant (Fig. 6). EGFP-YscN appeared as foci in both strains, indicating that YscO is dispensable for the assembly of EGFP-YscN. This suggests that, unlike the flagellar ATPase, the injectisome ATPase multimerizes in the absence of the FliJ orthologue. However, it should be kept in mind that assembly and multimerization of the ATPase may not be equivalent and that the similarity between YscO and FliJ is rather weak (Bennett and Hughes, 2000; Evans and Hughes, 2009).
Order of export of the early substrates
In the previous sections, we have determined at which stage the small early substrates are required to fulfil their function. We then wanted to determine the order of export. To this end, we monitored the export of the early substrates in an array of mutants affected individually in each of these substrates and other proteins with putative roles in the late steps of injectisome assembly (Fig. 7). Lack of YscF, YscI, YscO, YscX or YscY completely abolished the secretion of any tested substrate. In a yscP deletion strain, YscF and YscI, but no detectable amounts of any other protein were exported both under secretion-permissive conditions (Fig. 7A) and under secretion-restrictive conditions (Fig. 7B). Small amounts of YscF, YscI and YscP were also detected in the supernatant of a wild-type strain under secretion-restrictive conditions (Fig. 7B). Note that the amount of exported proteins under these secretion-restrictive conditions is significantly lower compared with a strain lacking the ‘Ca2+ plug’ protein YopN (Forsberg et al., 1991; Schubot et al., 2005), which exports proteins at a level comparable to the a wild-type strain under secretion-permissive conditions. Importantly, YscO or YscX were detected neither in the culture supernatant of yscP deletion strains, nor in the supernatant of a wild-type strain under restrictive conditions, suggesting that YscO and YscX are exported later than YscF and YscI. Notably, this does not contradict the fact that they are already required, inside the bacterium, for the export of YscI and YscF (see Fig. 5).
- Top of page
- Experimental procedures
- Supporting Information
In this study, we pursued our investigation of the assembly of the Yersinia Ysc injectisome. Our previous work had shown that the export apparatus (YscRSTUV) assembles in the IM and becomes embedded into the membrane ring scaffold (YscCDJ). This scaffold self-assembles inwards from the YscC secretin; the ATPase and C ring complex (YscKLNQ) then attaches to its cytosolic side (Diepold et al., 2010; 2011). The ultimate step is the formation of the needle by the newly formed export apparatus, embedded in the basal body. In this study, we showed that several small proteins, known to be required for T3S, play a role at this step. Besides the needle subunit YscF and its chaperones YscE and YscG (Day et al., 2000; Quinaud et al., 2005; 2007), these proteins are YscI, YscO, YscX and YscY. The three first ones were known to be exported proteins (Payne and Straley, 1998; Day and Plano, 2000; Kimbrough and Miller, 2000; Wood et al., 2008), while YscY was proposed to be the chaperone of YscX (Day and Plano, 2000). YscI is assumed to form an inner rod based on its relative similarity to PrgJ (Sukhan et al., 2003; Marlovits et al., 2004), but direct evidence is lacking. YscO has been proposed to be a chaperone escort (Evans et al., 2006) but its counterpart in the flagellum, FliJ, has also been shown to resemble the γ-subunit of the F1 ATPase and to promote hexamerization of the FliI ATPase (Ibuki et al., 2011).
We first observed that YscX and YscY are associated to YscV, in intact injectisomes as well as in the absence of any other protein. However, YscX and YscY required each other for this interaction. As we observed interaction between YscV and YscXY in conditions in which YscV does apparently not form multimers (i.e. in Yersinia enterocoliticaΔYscRSTU and in E. coli), we assume that every monomer of YscV binds YscXY and that YscV builds the multimer platform with YscX and YscY attached. We also observed that YscY-EGFP formed fluorescent spots, strongly suggesting that several copies of YscY are present at the injectisome. Although a YscV–YscY one-to-one stoichiometry seems to be the most logical interpretation of our data, and the observed fluorescence intensities are compatible with this, it needs to be confirmed. From all this, we conclude that YscX and YscY are structural components of the injectisome, belonging to the inner-most part of the export apparatus. The binding of YscX and YscY occurs independently of the recruitment of the ATPase and C ring since both proteins are present at the injectisome in the absence of YscN, which in turn is required for the formation of the C ring. Vice versa, the assembly of EGFP-YscQ does not depend on YscX or YscY (Diepold et al., 2010). All this suggests that YscX and YscY are recruited either together with YscV or immediately afterwards.
What could be the function of YscX and YscY? According to our results, they are not required for assembly of any part of the basal body, but are already needed for the first T3S-dependent export, the formation of the needle. YscX is a secreted protein and we showed that it does not belong to the group of early substrates because it is not exported in the absence of the ruler-switch protein YscP. Protein properties, genetic localization and binding data for YscY indicate that it could be a chaperone of the exported YscX (Day and Plano, 2000). However, various observations suggest that its function extends beyond the normal role of chaperones in the prevention of misfolding and mislocalization of its cargo protein (Page and Parsot, 2002). First, in contrast to all previously known chaperone binding partners, YscX exerts its function before it is exported. Second, YscY has been assigned an additional regulatory function, as it can bind to the class II chaperone SycD/LcrH (Francis et al., 2001; Broms et al., 2005). We would thus speculate that YscX and YscY, associated to YscV, contribute to determine the substrate specificity. Since we know that YscX is required for export of the early substrates (YscF, YscI, YscP), it might be released afterwards, allowing YscV–YscY to recognize the next category of substrates, the translocators, which would bring them in a ‘pole position’ for the export, once contact to a host cell has been established. YscY largely consists of TPR repeats, a motif often seen in class II and class III chaperones, which also specifically recognize substrates (Page and Parsot, 2002; Pallen et al., 2003; Quinaud et al., 2007). In support of the idea that YscXY contribute to substrate recognition, a structural homology search revealed that the two closest bacterial homologues of YscY are PcrH from Pseudomonas and IpgC from Shigella, which are both chaperones of the hydrophobic T3S translocator proteins (Soding, 2005; Bordoli et al., 2009; Lunelli et al., 2009; Job et al., 2010). The long-known interaction between SycD and YscY could therefore facilitate the hand-over of substrates between the two proteins. However, so far, no direct interaction of YscY with the translocators has been observed and no mutant of YscX or YscY with a specific effect on substrate specificity has been isolated.
A similar role of the export apparatus in substrate selection has recently been discovered in the flagellum. Bange et al. showed that the flagellar YscV homologue FlhA binds FliD and FliT, the chaperones of flagellin and of the cap respectively. Almost the whole FlhA protein is required for this binding, suggesting a close interaction (Bange et al., 2010). The authors proposed that the number of FliT chaperones bound to FlhA could determine the number of exported cap substrates. Although the stoichiometry of YscV and the resulting number of binding sites for YscY is still unclear, it appears possible that the number of export sites for the hydrophobic translocators could be determined in a similar fashion.
Clear homologues to YscX and YscY are present in the various members of the Ysc family of T3S systems (Troisfontaines and Cornelis, 2005), namely Pcr3/4 in Pseudomonas aeruginosa (Broms et al., 2005), AscX/Y in Aeromonas spp. (Burr et al., 2002), SctX/Y in Photorhabdus spp. (Waterfield et al., 2002) and VscX/Y in Vibrio spp. (Makino et al., 2003). Interestingly, Pcr3 and Pcr4 from P. aeruginosa behave differently from YscXY (Yang et al., 2007) in the sense that Pcr3 (YscX) is not exported while Pcr4 (YscY) is. Its export is enhanced under secretion-restrictive conditions, suggesting that it is exported before the effectors. Yang et al. found an interaction between Pcr1 (TyeA) and Pcr4 (YscY), giving a possible link to control of effector secretion (Forsberg et al., 1991; Iriarte and Cornelis, 1999; Ferracci et al., 2005; Schubot et al., 2005). However, Pcr1 (TyeA), Pcr2 (SycN) and Pcr4 (YscY) were all exported even in conditions that are non-permissive for secretion (presence of Ca2+) (Yang et al., 2007). Although no homologues of YscX or YscY have been found so far outside the Ysc family of T3S (Broms et al., 2005; Yang et al., 2007), we expect that functional homologues may exist in other systems: since the main interaction partner YscV is conserved across its whole length in all compared species (Fig. S3), it is unlikely that this protein alone could take over the role of YscX and YscY in other systems.
YscO is another small exported protein that is essential for T3S. Based on synteny, protein size and predicted structure, its most likely homologue in the flagellum is FliJ, but there is no significant sequence similarity (Payne and Straley, 1998; Evans and Hughes, 2009). FliJ was shown to be an escort chaperone, specifically recruiting some empty chaperones to the ATPase complex. Since FliJ has less affinity for the chaperone than the usual partner of the chaperone, FliJ was proposed to recycle empty chaperones to their cognate binding partners (Evans et al., 2006). Based on this observation and the influence of FliJ on the binding of chaperones to the export apparatus, a role in substrate selection was proposed (Bange et al., 2010). Like FliJ, YscO was shown to bind empty chaperones, notably SycD (Evans and Hughes, 2009), the chaperone of translocators (Wattiau et al., 1994) and hence, it could play a similar role as FliJ. However, FliJ has also been shown to promote multimerization of the ATPase (Ibuki et al., 2011) and since YscO is encoded just besides YscN, we tested whether YscO would be required for the multimerization of EGFP-YscN. Since it was not, the escort chaperone hypothesis remains the most likely to explain YscO's function.
Finally, we investigated the export of the reporter T3S substrate YscP1–137-PhoA into the periplasm by the apparatus missing either of the small soluble components YscF, YscI, YscO or YscX. We observed that YscP1–137-PhoA was not exported in the absence of YscI, YscO or YscX, reinforcing the earlier observations that these three proteins are essential for export. However, interestingly, in the absence of YscF, YscP1–137-PhoA accumulated in the periplasm, suggesting that YscF is not required for the export apparatus to be functional but that its polymerization opens the plug in the secretin YscC. To our knowledge, this is the very first report showing a different behaviour of yscF and yscI deletion mutants.
Taken together, our results describe the order of events prior to the final formation of the T3S needle. Importantly, we discovered that two so far little-characterized proteins, YscX and YscY, strongly and directly bind to the cytosolic domain of YscV. Like YscV, YscX and YscY are present at the basis of the apparatus in multiple copies. Since the cytosolic domains of YscV homologues have been shown to form a prominent ‘export platform’ below the proposed export gate (Chen et al., 2011), and these domains are involved in substrate selection (Bange et al., 2010), YscX and YscY are likely candidates to play a role in the binding of export substrates to the export apparatus, which is completely compatible with the structural prediction for YscY. Our results also showed that YscO is not needed for the YscN ATPase to assemble, that YscX and YscO are only exported after the substrate specificity switch has occurred and, finally, suggest that YscF is the protein that opens the secretin gate.
- Top of page
- Experimental procedures
- Supporting Information
Bacterial strains, plasmids and genetic constructions
Strains and constructs used in the experiments are shown in Table 3; all bacterial strains, plasmids and genetic constructions are given in Table S1. E. coli Top10 and BW19610 were used for cloning and E. coli SM10 λ pir+ for conjugation. E. coli strains were grown routinely on Luria–Bertani (LB) agar plates or in liquid LB medium at 37°C. Ampicillin and Streptomycin were used at concentrations of 200 µg ml−1 and 100 µg ml−1 to select for pBAD vectors and suicide vectors. Y. enterocolitica strains were routinely grown at 25°C in brain heart infusion (BHI) broth containing 35 µg ml−1 nalidixic acid. Plasmids were generated using Phusion polymerase (Finnzymes, Espoo, Finland). Mutators for modification or deletion of genes in the pYV plasmids were constructed as described in Diepold et al. (2011). All constructs were confirmed by sequencing (Microsynth, St. Gallen, Switzerland).
|pYV plasmids and Y. enterocolitica strains|
|pYV plasmid||Relevant characteristics||Strain||References|
|pAD4037||pYVe40 yscVΔ5–680||E40||Diepold et al. (2010)|
|pAD4136||pYVe40 yscNΔ2–427||E40||Diepold et al. (2010)|
|pAD4160||pYVe40 yscVΔ5–680ΔyscJ||E40||Diepold et al. (2011)|
|pAD4161||pYVe40 yscVΔ5–680 yscNΔ2–427||E40||Diepold et al. (2011)|
|pAD4168||pYVe40 yscVΔ5–680 yscCΔ2–598||E40||Diepold et al. (2011)|
|pAD4169||pYVe40 yscVΔ5–680 yscDΔ2–404||E40||Diepold et al. (2011)|
|pAD4173||pYVe40 yscV-EGFP||E40||Diepold et al. (2011)|
|pAD4186||pYVe40 yscVΔ5–680ΔyscRSTU||E40||Diepold et al. (2011)|
|pAD4191||pYVe40 yscVΔ5–680 yscXΔ42–75 (pIM405 mutated with pAD134)||E40||This study|
|pAD4192||pYVe40 yscVΔ5–680 yscYΔ21–45 (pIM406 mutated with pAD134)||E40||This study|
|pAD4196||pYVe40 yscV-EGFP yscXΔ42–75||E40||Diepold et al. (2011)|
|pAD4202||pYVe40 yscV-EGFP yscYΔ21–45 (pIM406 mutated with pAD208)||E40||This study|
|pADSN4212||pYVe40 yscNOΔYscN2-YscO149 (pYVe40 mutated with pADSN268)||E40||This study|
|pIM405||pYVe40 yscXΔ42–75||E40||Iriarte and Cornelis (1999)|
|pIM406||pYVe40 yscYΔ21–45||E40||Iriarte and Cornelis (1999)|
|pIM41||pYVe40 yopN45 (does not encode YopN)||E40||Boland et al. (1996)|
|pISO4006||pYVe40 yscFΔ1–74||E40||Diepold et al. (2010)|
|pISO4008||pYVe40 yscOΔ4–149||E40||Diepold et al. (2010)|
|pKEM4001||pYVe40 ΔyscI||E40||Diepold et al. (2010)|
|pLJ4036||pYVe40 ΔyscP||E40||Agrain et al. (2005a)|
|pUW22711||pYVe227 yscY-EGFP yscVΔ158–438 (pMRS2276 mutated with pUW025)||W22703||This study|
|pUW4007||pYVe40 yscY-EGFP (pYVe40 mutated with pUW025)||E40||This study|
|pUW4008||pYVe40 yscY-EGFP yscXΔ42–75 (pIM405 mutated with pUW025)||E40||This study|
|pUW4009||pYVe40 yscY-EGFP yscFΔ1–74 (pISO4006 mutated with pUW025)||E40||This study|
|pUW4010||pYVe40 yscY-EGFP yscNΔ2–427 (pAD4136 mutated with pUW025)||E40||This study|
|pYVe40||Wild-type pYV plasmid of Y. enterocolitica E40||E40 (WT)||Sory et al. (1995)|
|Expression plasmids, vectors and mutators|
|pAD148||pGEX-6P-1::yscV322–704 (GST-PreScission-YscV322–704)||This study|
|pAD153||pBAD::yscV||Diepold et al. (2011)|
|pAD172||pGEX-6P-1::yscV356–704 (GST-PreScission-YscV356–704)||This study|
|pAD173||pGEX-6P-1::yscV412–704 (GST-PreScission-YscV412–704)||This study|
|pAD182||pBAD::egfp-yscN||Diepold et al. (2010)|
|pAD201||pBAD::yscV-his-flag||Diepold et al. (2011)|
|pAD227||pBAD::yscV1–331-his-flag||Diepold et al. (2011)|
|pAD235||pGEX-6P-1::yscV322–445 (GST-PreScission-YscV322–445)||This study|
|pAD237||pGEX-6P-1::yscV322–511 (GST-PreScission-YscV322–511)||This study|
|pAD238||pGEX-6P-1::yscV322–601 (GST-PreScission-YscV322–601)||This study|
|pAD267||pGEX-6P-1::yscV441–704 (GST-PreScission-YscV441–704)||This study|
|pGEX-6P-1||High-copy expression vector for N-terminal GST-PreScission fusions||GE Healthcare|
Yersinia enterocolitica mutants were generated by allelic exchange, replacing the wild-type gene on the virulence plasmid by the mutated version. Completion of the allelic exchange was tested for by plating diploid bacteria on plates containing 5% sucrose (Kaniga et al., 1991).
Y. enterocolitica cultures for T3S and microscopy analysis
Cultures were inoculated to an optical density at 600 nm (OD600) of 0.12 in BHI broth containing sodium oxalate (20 mM) (BHI-Ox, secretion-permissive permissions) or CaCl2 (5 mM) (BHI-Ca2+, secretion-restrictive conditions) supplemented with glycerol (4 mg ml−1) and MgCl2 (20 mM). After 1.5 h of growth at 25°C, induction of the yop regulon was performed by shifting the culture to 37°C. Expression of the pBAD constructs was induced by adding 0.03–0.3% l-arabinose to the culture just before the shift to 37°C. After 3 h of incubation at 37°C, cultures were used for further analysis.
Secretion analysis and immunoblotting
Bacteria and supernatant (SN) fractions were separated by centrifugation at 20 800 g for 10 min at 4°C. The cell pellet was taken as total cell (TC) fraction. Proteins in the supernatant were precipitated with trichloroacetic acid 10% (w/v) final for 1 h at 4°C. Supernatant and total cell fractions were separated on 12% or 15% SDS-PAGE gels respectively. Unless mentioned otherwise, proteins secreted by 3 × 108 bacteria (SN) or produced by 1 × 108 bacteria (TC) were loaded per lane. Immunoblotting was carried out using rabbit, goat (only for GST) or rat (only for YopB) polyclonal antibodies against GFP (Invitrogen; 1:800), GST (Amersham Bioscience; 1:500), LcrV (MIPA220; 1:1000), PhoA (MIPA7; 1:1000), YopB (MIPA88; 1:500), YopP (MIPA95; 1:1000), YscF (MIPA223; 1:1000), YscI (MIPA83; 1:500), YscO (MIPA65; 1:1000), YscP (MIPA57; 1:3000) and YscX (MIPA224; 1:500). Detection was performed with swine anti-rabbit (Dako; 1:5000), rabbit anti-goat (Dako; 1:5000) or goat anti-rat (SouthernBiotech; 1:5000) secondary antibodies conjugated to horseradish peroxidase, before development with LumiGLO Reserve chemiluminescent substrate (KPL).
For fluorescence imaging, about 2 µl of bacterial culture (see above) was placed on a microscope slide layered with a pad of 2% agarose in PBS. A Deltavision Spectris optical sectioning microscope (Applied Precision, Issaquah, WA, USA) equipped with a UPlanSApo 100×/1.40 oil objective (Olympus, Tokyo, Japan) and a coolSNAP HQ CCD camera (Photometrics, Tucson, AZ, USA) was used to take differential interference contrast (DIC) and fluorescence photomicrographs. GFP filter sets (Ex 490/20 nm, Em 525/30 nm) were used for GFP visualization. DIC frames were taken with 0.1 s and fluorescence frames with 1.0 s exposure time. Per image, a Z-stack containing 20 frames per wavelength with a spacing of 150 nm was acquired. The stacks were deconvolved using softWoRx v3.3.6 with standard settings (Applied Precision, WA). A representative DIC frame and the corresponding fluorescence frame were selected and further processed with the ImageJ software.
Pull-down assay, co-immunoprecipitation and mass spectrometry
Pull-down assays of GST-YscVC, YscX and YscY were performed in E. coli BL21(DE3). YscX and YscY were expressed from a pCDFduet vector; the various N-terminally GST-tagged versions of YscV were expressed from vector pGEX-6P-1 and induced by addition of 0.3 mM IPTG at an OD600 of 0.6–0.8. The culture was incubated at 25°C for 5 h, pelleted (10 min, 8000 g, 4°C) and resuspended in 1/15 original culture volume (OCV) of lysis buffer [phosphate buffered saline (PBS) + 5 mM DTT]. The solution was frozen at −80°C, thawed and lysed by passage through a French pressure cell. At this point, the protein content of the lysate was determined by a Bradford assay (Bradford, 1976). After centrifugation (15 500 g, 36 min, 4°C), the supernatant was passed through 0.45 µm filters and 1 ml of pre-washed glutathione-agarose/300 ml original culture was added. The mixture was incubated for 90 min at 4°C on a rotating wheel. The following centrifugations were performed (400 g, 30 s, 4°C): removal of the unbound supernatant; three wash steps with 1/30 OCV of lysis buffer; 15 min incubation with 1/300 OCV elution buffer (PBS + 10 mM reduced glutathione), two more elution steps with 1/300 OCV elution buffer. The pooled elution fractions were precipitated, normalized for the protein content after lysis and analysed on a 15% SDS-PAGE gel.
Co-immunoprecipitation of injectisome complexes in Y. enterocolitica was performed as described (Diepold et al., 2010; 2011). The complete eluate was pooled and centrifuged for 5 min at 5000 g to completely remove resin. The eluate corresponding to the purified proteins from 100 ml initial bacterial culture was precipitated for 1 h with 10% v/v trichloroacetic acid and analysed by mass spectrometry as described by Manfredi et al. (2011). Scaffold 3.2.0 (Proteome Software, Portland, OR, USA) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95% probability as specified by the Peptide Prophet algorithm (Keller et al., 2002). Protein identifications were accepted if they could be established at greater than 90% probability and contained at least two identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii et al., 2003). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony.
Alkaline phosphatase assay
The alkaline phosphatase assay was performed as described by Manoil and Beckwith (1986), modified as follows: cultures were inoculated to an OD600 of 0.18 under secretion-permissive conditions (BHI containing 20 mM sodium oxalate, 4 mg ml−1 glycerol, 20 mM MgCl2). After 70 min of growth at 25°C, the yop regulon was induced by shifting the culture to 37°C. Expression of the alkaline phosphatase reporter construct was induced by adding 0.3% l-arabinose to the culture just before the shift to 37°C. After 2 h of incubation at 37°C, the optical density of the cultures were measured and 8 × 108 bacteria (1.6 ODu) were pelleted (4000 g, 5 min, 37°C). The bacteria were carefully resuspended in 0.5 ml of pre-warmed resuspension buffer (0.1 M Tris-HCl pH 7.8, 5 mM EDTA) and incubated for 15 min at 37°C. They were then permeabilized by addition of 20 µl of chloroform and 20 µl of 0.075% SDS, vortexed for 10 s, and incubated at 37°C. After 15 min, 500 µl of pNPP buffer (20 mM para-nitrophenylphosphate, 1 M Tris-HCl pH 9.0, 10 mM MgCl2, 10 mM CaCl2) was added and the solution was incubated until colour change could be observed. The reaction was then stopped by the addition of 500 µl of 2 M NaOH. To remove cells, the mixture was pelleted for 2 min at 20 800 g. The supernatant was removed and the optical density at 410 nm was measured in triplicates. The alkaline phosphatase activity was expressed in mOD420 per minute per OD600 of the corresponding bacterial culture (Brickman and Beckwith, 1975). Analysis of the supernatant by immunoblot was performed after 2 h of incubation at 37°C with secreted proteins from 6 × 108 bacteria.
- Top of page
- Experimental procedures
- Supporting Information
This work was supported by the Swiss National Science Foundation (Grant 3100A0-128659).
- Top of page
- Experimental procedures
- Supporting Information
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